Collagen is the most abundant protein found in vertebrates and serves as a structural protein of tissues. The several types of collagen are a family of genetically related proteins that exhibit fundamentally similar secondary and tertiary protein structures. Type I Collagen is the most prevalent and is found, for example, in skin, tendon and bone.
Collagenolytic serine protease 1 (EC 2.4.21.32) isolated from the hepatopancreas of the fiddler crab, Uca pugilator, is the first known serine protease capable of cleaving native type I triple helical collagen. Serine collagenases have since been isolated from a variety of organisms, and are thought to be primarily involved in the digestion of foodstuffs. In addition to its eponymous activity, crab collagenase also possesses significant trypsin, chymotrypsin, and elastase-like substrate specificities, and is the most efficient serine protease known in the hydrolysis of P1-Gln and P1-Leu amide substrates. Preferences in the cleavage of peptide bonds within a relaxed domain of collagen mirrors the enzymes' specificity toward small peptidyl substrates. The ability of collagenase, but not trypsin, chymotrypsin or other homologs, to cleave triple helical collagen arises from unique extended substrate binding sites in collagenase.
In 1980 an amino acid sequence was published that was said to be of fiddler crab collagenase (Grant, et al. Biochemistry, 19 pp. 4653-4659). The postulated amino acid sequence of fiddler crab collagenase identified it as a member of the chymotrypsin-like serine proteases. However, as will be described hereinafter, this published amino acid sequence was incorrect.
U.S. Pat. No. 5,143,837, inventors Sova et al., issued Sep. 1, 1992, describes an enzyme complex derived from crabs. This complex is said to be useful to cleave standard synthetic and protein substrates and to possess a chymotrypsin trypsin and elastase-like activity; however, complexes such as the complex described by Sova et al., have variable properties, particularly from batch to batch. That is, such complexes may include multiple forms of collagenase, and contaminating non-collagenase proteases. Such variability complicates efforts to use the desired collagenase. This tends to be particularly frustrating for researchers and to persons involved in tissue culture, where batch to batch variations can, at best, create difficulties for use. Thus, a reliably pure, homogenous collagenase would be desirable.
U.S. Pat. No. 5,418,157, issued May 23, 1995, inventors Lin et al., describe genetically engineered E. coli said to carry vectors containing inserts that code for Clostridium histolyticum collagenase, having a molecular weight of about 68,000 Daltons. However, this collagenase is considerably larger than crab collagenase and is also far less specific. The collagenase described by Patent '157 is less specific than fiddler crab collagenase due to its evolution as a non-specific enzyme serving a nutritive role for the soil bacterium that produces it. Thus, the bacterial collagenase degrades any protein material with which it comes into contact. Its degradation results in amino acids and the organism follows the resulting amino acid gradient produced for nutritive purposes. Collagen is just one of many proteins that the bacterial collagenase degrades, and so it does so in a very non-specific fashion.